Project Summary Sporadic neurodegenerative diseases (NDD) are frequent, heterogeneous and cause severe disabilities in ~30 millions patients worldwide. They represent a major and growing public health challenge, as their causes are unclear and therapeutic options are few. Genetic susceptibility alleles have been identified in a proportion of patients but population-based genetic, transcriptomic, and epigenetic studies have so far provided few significant mechanistic breakthroughs translatable into diagnostic and therapeutic strategies. Among environmental factors it is noted that NDD incidence increases after traumatic brain injury. Nevertheless, the molecular mechanism(s) that underlie neuronal dysfunction and death in these diseases are unclear, which impedes the development of effective treatments. Chronic activation of microglia, the resident macrophage of the brain, is observed at sites of neuronal damage and mutations affecting the function of microglia have been identified in (rare) inherited recessive and dominant neurodegenerative diseases. However, microglia activation in NDD is believed to be in general a reactive process, and its actual pathogenic role has been difficult to resolve in genetic or molecular terms. Despite the important roles of post-zygotic somatic DNA mutations in developmental and tumoral diseases, the possibility that neurodegeneration may be due to deleterious microglia somatic mutants has not been investigated, in large part because of technical limitations, as microglia only represent 5% of brain cells, and develop and maintain locally in the brain. We hypothesize that microglia clonal heterogeneity (mosaicism) is widespread in the human brain and that somatic variants, such as `cancer' mutations (e.g. Kinases gain-of-function) that confer proliferative/activation advantage to microglial clones cause or contribute to neurodegeneration in patients. Our published results from reverse genetic experiments in mice strongly support this hypothesis, and we have obtained preliminary forward genetic evidence that candidate pathogenic clones can be identified in human patients. We have developed protocols to isolate and sequence at high depth microglia nuclei in human brains. Support through the NIH Director's Transformative Research Award at this stage is absolutely needed to allow us to characterize pathogenic clones in a representative sample among the heterogeneous spectrum of NDD, which is an essential task to frame the role of microglia clonality in the neurodegenerative process, and will make possible future studies that will likely focus on specific subsets of patients or on recurrent deleterious clonal events. Results from our project will radically transform our molecular understanding of NDD and provide novel molecular diagnosis in subsets of patients. Because small molecule inhibitors for many of the genes and mutations that confer clonal advantage have been developed by the field of cancer biology, our project should also result in the development of targeted therapies for subsets of NDD.